N-Myristoylation refers to the co-translational addition of myristic acid to the N-terminal glycine of many viral and eukaryotic proteins which are essential to normal cell functioning and/or are potential therapeutic targets. Covalently bound myristate is required for the proper subcellular targeting and cell functions of Src family tyrosine protein kinases such as pp6O(src) and p56(lck) and transmembrane heterotrimeric G proteins, the membrane association and assembly of Gag polyprotein precursors of a number of retroviruses, and the proper folding of capsid proteins of papovaviruses and picornaviruses. It is also required for the transforming activity of pp60(v-src). We have purified the enzyme catalyzing N-myristoylation, N-myristoyltransferase (NMT), from bovine brain and shown it to consist of a mixture of two interconvertible high molecular weight multimers made up of 6O kDa NMT subunits. Also found are small amounts of a fully active NMT monomer which arises by proteolytic removal of the 12 kDa N-terminus of the 60 kDa subunit. NMT specific activities are also found to vary significantly among cells and tissues with highest activity to date in human lymphoid cell lines such as Molt 4 and CEM. Subcellular fractionation of CEM cells revealed NMT activity exclusively in the ribosomal fraction. Immunoblotting with a human NMT peptide antibody visualized a 57 kDa NMT subunit in the ribosomal pellet which was rapidly converted into a 48 kDa form when stored in the absence of proteolytic inhibitors. These data are consistent with the specific targeting of NMT to subcellular targets where it can efficiently N- myristoylate substrates during protein synthesis. A unique requirement for N-myristoylation for the viability of lymphoid cells is suggested by the selective sensitivity of a subpannel of 6 leukemic/lymphoid cell lines to two inhibitors of NMT, 1-bromo-2-pentadecanone and myristoyl hydroxymate. Both compounds selectively block the in vivo N-myristoylation of several prominent low molecular weight proteins in CEM cells. The data reveal a previously unappreciated level of NMT complexity which may have important mechanistic, regulatory, and/or therapeutic significance for N- myristoylation in leukemic/lymphoid cells.